Method of preventing wrong color formation of n-(carboxymethylaminocarbony)-4,4′-bis(dimethylamino) diphenylamine sodium, reagent solution for the method, and measurement method employing the method

ABSTRACT

The present invention provides a method of preventing erroneous color development of N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt as a color-developing substrate, thereby improving the accuracy of measurement utilizing a redox reaction performed using the color-developing substrate. A tetrazolium compound, sodium azide, and the color-developing substrate are added to a sample in the presence of a surfactant. A reaction between an oxidizing substance derived from an analyte in the sample and the color-developing substrate, which develops color by oxidation, is caused by an oxidoreductase. By measuring the color developed, the amount of the oxidizing substance is determined. The concentrations of the respective components in the reaction solution are set so that 0.01 to 1 mmol of the tetrazolium compound, 0.003 to 0.5 mmol of the sodium azide, and 0.006 to 0.4 mmol of the surfactant are present per μmol of the color-developing substrate, and the pH of the reaction solution is set in the range from 6 to 9.

This application is a 371 of PCT/JP03/05642 filed May 2, 2003.

TECHNICAL FIELD

The present invention relates to a method of preventing erroneous colordevelopment ofN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt, and to a reagent solution and a measurement methodutilizing a redox reaction using the method.

BACKGROUND ART

Conventionally, the measurement of the amount of an analyte in a sampleusing a redox reaction has been utilized for a wide range ofapplications. For example, such measurement is carried out in thefollowing manner. First, a peroxidase (hereinafter referred to as “POD”)and a reducing agent are added to an oxidizing substance as an analyteor to an oxidizing substance formed from an analyte, so that a redoxreaction occurs between the oxidizing substance and the reducing agentwith the POD as a catalyst. When a reducing agent that develops colorwhen it is oxidized is used as the reducing agent, the amount of theoxidizing substance can be determined by measuring the color developedbecause there is a correlation between the amount of the color developedand the amount of the oxidizing substance. As the reducing agent thatdevelops color when it is oxidized,N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt has been used.

However, such a method does not exhibit sufficient measurementsensitivity and thus may fail to improve the accuracy of themeasurement.

DISCLOSURE OF INVENTION

In light of the above-described problem, the inventors of the presentinvention conducted in-depth researches and found out that themeasurement sensitivity can be improved by carrying out the redoxreaction in the presence of a tetrazolium compound and sodium azide.This finding was already filed as another patent application. However,the inventors of the present invention further found that, although sucha measurement method can improve the measurement sensitivity asdescribed above, it brings about another problem that erroneous colordevelopment ofN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt as a substrate that develops color (hereinafter referred toas a “color-developing substrate”) may occur prior to the redox reactiondue to the presence of theN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt, the tetrazolium compound, and the sodium azide together inthe solution. Such erroneous color development leads to an increase inbackground in the above-described measurement of the color developedand, in some cases, to a shortage of the color-developing substrate inthe redox reaction even though the amount of the color-developingsubstrate added is sufficient.

Therefore, it is an object of the present invention to provide a methodof preventing erroneous color development of N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylamine sodium salt.

In order to achieve the above object, the present invention provides amethod of preventing erroneous color development ofN-(carboxymethylamino carbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt (hereinafter also referred to as “DA-64”) in an aqueoussolvent containing a tetrazolium compound and sodium azide, including:mixing a tetrazolium compound, sodium azide, and DA-64 in an aqueoussolvent in the presence of a surfactant, wherein 0.01 to 1 mmol of thetetrazolium compound, 0.003 to 0.5 mmol of the sodium azide, and 0.006to 0.4 mmol of the surfactant are present per μmol of the DA-64, and apH of the aqueous solvent containing the DA-64, the tetrazoliumcompound, the sodium azide, and the surfactant is in a range from 6 to9.

By mixing the above-described three components in the presence of thesurfactant and setting the concentrations of the respective componentsand the pH of the mixture in the above-described ranges, theabove-described erroneous color development of the DA-64 can besuppressed even though the “DA-64, tetrazolium compound, and sodiumazide” are present together in the aqueous solvent. Thus, if such amethod of preventing the erroneous color development is applied to ameasurement using a redox reaction described later, an increase inbackground absorbance in the measurement of an absorbance can besuppressed so that an analyte can be measured with high accuracy. Theorder of adding the respective components is not particularly limited aslong as the surfactant is present when all of the DA-64, the tetrazoliumcompound, and the sodium azide are mixed with each other in the aqueoussolvent. Thus, for example, the surfactant may be added to the aqueoussolvent in advance and then the remaining three components may be addedto the aqueous solvent, separately. Alternatively, after the tetrazoliumcompound and the sodium azide have been added to the aqueous solvent,the DA-64 may be added in the presence of the surfactant. These examplesare for illustration only, and the order of adding the components is notlimited to these examples.

Next, the present invention provides a DA-64 reagent solution includingDA-64, a tetrazolium compound, sodium azide, and a surfactant, wherein0.01 to 1 mmol of the tetrazolium compound, 0.003 to 0.5 mmol of thesodium azide, and 0.006 to 0.4 mmol of the surfactant are present perμmol of the DA-64, and a pH of the reagent solution is in a range from 6to 9.

In the reagent solution having such a composition, the erroneous colordevelopment of the DA-64 also can be prevented even in the presence ofthe tetrazolium compound and the sodium azide, as in the case of theabove-described method of preventing the erroneous color development.Thus, it becomes possible to store DA-64 stably in the form of asolution, and such a reagent solution suitably is used for a measurementmethod described below and the like, for example.

Next, the present invention provides a method of measuring an oxidizingsubstance derived from an analyte in a sample, including: causing areaction between the oxidizing substance derived from the analyte andDA-64 as a color-developing substrate, the reaction being caused by anoxidoreductase in the presence of a tetrazolium compound and sodiumazide; and determining an amount of the oxidizing substance by measuringthe color developed by the color-developing substrate. In this method,the tetrazolium compound, the sodium azide, and the color-developingsubstrate are mixed in an aqueous solvent in the presence of asurfactant so that 0.01 to 1 mmol of the tetrazolium compound, 0.003 to0.5 mmol of the sodium azide, and 0.006 to 0.4 mmol of the surfactantare present per μmol of the DA-64 as the color-developing substrate, anda pH of the resultant mixture is in a range from 6 to 9.

Such a measurement method is excellent not only in measurementsensitivity but also in measurement accuracy because an increase inbackground due to the erroneous color development of the DA-64 can besuppressed. In the present invention, “an oxidizing substance derivedfrom an analyte” includes the analyte itself, an oxidizing substancecontained in the analyte, and an oxidizing substance formed from theanalyte using an oxidoreductase or the like.

In the measurement method according to the present invention, theaqueous solvent preferably is a sample solution containing the analyte.For example, it is preferable that the tetrazolium compound and thesodium azide are added to the sample solution and thereafter, theN-(carboxymethylamino carbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt (DA-64) and the oxidoreductase further are added to thesample solution in the presence of the surfactant to cause the reactionby the oxidoreductase.

In the measurement method according to present invention, the type ofthe sample is not particularly limited. The method also can be appliedto samples other than whole blood, plasma, serum, and blood cells, e.g.,biological samples such as urine and spinal fluid, drinks such asjuices, and foods such as soy sauce and Worcestershire sauce.

In the measurement method according to present invention, the analyte isnot particularly limited, as long as a redox reaction is utilized. Forexample, the analyte may be components in whole blood, components inerythrocytes, components in plasma, components in serum, components inurine, components in spinal fluid, and the like, and it preferably is acomponent in erythrocytes. For example, when a component in erythrocytesis to be measured, whole blood itself may be hemolyzed to prepare asample, or erythrocytes may be separated from whole blood and hemolyzedto prepare a sample. Specific examples of the analyte include glycatedproteins such as glycated hemoglobin and glycated albumin, glycatedpeptides, glycated amino acids, glucose, uric acid, cholesterol,creatinine, sarcosine, and glycerol. Among these, glycated proteins aremore preferable and glycated hemoglobin is particularly preferable. Thereason for this is as follows. Glycated hemoglobin has been regarded asan important indicator in the diagnosis, therapy, and the like ofdiabetes, because it reflects the patient's past history of bloodglucose levels. Since the amount of glycated hemoglobin can be measuredaccurately according to the present invention, the reliability ofglycated hemoglobin as the indicator is improved. Thus, the presentinvention is beneficial in the field of clinical medicine and the like.

In the measurement method of the present invention, when the analyte isa glycated protein, it is preferable that a glycation site thereof isdegraded by oxidation with a fructosyl amino acid oxidase (hereinafterreferred to as “FAOD”) so that hydrogen peroxide is formed. Also, whenthe analyte is a glycated peptide or a glycated amino acid, it ispreferable that the glycated peptide or the glycated amino acidsimilarly is subjected to the action of a FAOD. The hydrogen peroxidethus formed corresponds to the above-described oxidizing substancederived from the analyte. Moreover, it is preferable that glycatedproteins and glycated peptides are treated with a protease prior to theFAOD treatment as necessary.

As the FAOD, a FAOD catalyzing a reaction represented by Formula (1)below preferably is used.R¹—CO—CH₂—NH—R²+H₂O+O₂→R¹—CO—CHO+NH₂—R²+H₂O₂  (1)

In Formula (1), R¹ denotes a hydroxyl group or a residue derived fromthe sugar before glycation (i.e., sugar residue). The sugar residue (R¹)is an aldose residue when the sugar before glycation is aldose, and is aketose residue when the sugar before glycation is ketose. For example,when the sugar before glycation is glucose, it takes a fructosestructure after glycation by an Amadori rearrangement. In this case, thesugar residue (R¹) becomes a glucose residue (an aldose residue). Thissugar residue (R¹) can be represented, for example, by—[CH(OH)]_(n)—CH₂OHwhere n is an integer of 0 to 6.

In Formula (1), R² is not particularly limited. However, when thesubstrate is a glycated amino acid, a glycated peptide, or a glycatedprotein, for example, there is a difference between the case where anα-amino group is glycated and the case where an amino group other thanthe α-amino group is glycated.

In Formula (1), when an α-amino group is glycated, R² is an amino acidresidue or a peptide residue represented by Formula (2) below.—CHR³—CO —R⁴  (2)

In Formula (2), R³ denotes an amino-acid side chain group. R⁴ denotes ahydroxyl group, an amino acid residue, or a peptide residue, and can berepresented, for example, by Formula (3) below. In Formula (3), n is aninteger of 0 or more, and R³ denotes an amino-acid side chain group asin the above.—(NH—CHR³—CO)_(n)—OH  (3)

In Formula (1), when an amino group other than the α-amino group isglycated (i.e., an amino-acid side chain group is glycated), R² can berepresented by Formula (4) below.—R⁵—CH(NH—R⁶)—CO—R⁷  (4)

In Formula (4), R⁵ denotes a portion other than the glycated amino groupin the amino-acid side chain group. For example, when the glycated aminoacid is lysine, R⁵ is as follows.—CH₂—CH₂—CH₂—CH₂—For another example, when the glycated amino acid is arginine, R⁵ is asfollows.—CH₂—CH₂—CH₂—NH—CH(NH₂)—

In Formula (4), R⁶ denotes hydrogen, an amino acid residue, or a peptideresidue, and can be represented, for example, by Formula (5) below. InFormula (5), n denotes an integer of 0 or more, and R³ denotes anamino-acid side chain group as in the above.—(CO—CHR³—NH)_(n)—H  (5)

In Formula (4), R⁷ denotes a hydroxyl group, an amino acid residue, or apeptide residue, and can be represented, for example, by Formula (6)below. In Formula (6), n is an integer of 0 or more, and R³ denotes anamino-acid side chain group as in the above.—(NH—CHR³—CO)_(n)—OH  (6)

As the FAOD, a commercially available product named Fructosyl-Amino AcidOxidase (FAOX-E) (manufactured by Kikkoman Corporation) specific for aglycated amino acid having a glycated α-amino group, commerciallyavailable products named FOD (manufactured by Asahi Chemical IndustryCo., Ltd.) and KAO (manufactured by Genzyme Japan K.K.) specific for aglycated amino acid having a glycated α-amino group and for a glycatedamino acid such as lysine having a glycated ε-amino group, and the likemay be used, for example.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the change in absorbance with time in oneexample of a measurement method according to the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention prevents the erroneous color development of DA-64in the following manner, for example.

First, a surfactant is dissolved in an aqueous solvent, and then atetrazolium compound, sodium azide, and DA-64 are mixed with theresultant solution. The tetrazolium compound, the sodium azide, and theDA-64 are mixed so that 0.01 to 1 mmol of the tetrazolium compound,0.003 to 0.5 mmol of the sodium azide, and 0.006 to 0.4 mmol of thesurfactant are present per μmol of the DA-64, and the pH of the mixtureis adjusted so as to be in the range from 6 to 9.

Preferably, 0.02 to 0.8 mmol of the tetrazolium compound, 0.01 to 0.3mmol of the sodium azide, and 0.01 to 0.4 mmol of the surfactant arepresent per μmol of the DA-64. Particularly preferably, 0.03 to 0.6 mmolof the tetrazolium compound, 0.01 to 0.2 mmol of the sodium azide, and0.04 to 0.3 mmol of the surfactant are present per μmol of the DA-64.Furthermore, the pH of the mixture preferably is in the range from 6 to9, more preferably from 6.5 to 8.

As described above, the order of adding the respective components is notparticularly limited, as long as the surfactant falling within theabove-described concentration range is present when mixing the remainingthree components: the tetrazolium compound, the sodium azide, and theDA-64.

The type of the aqueous solvent is not particularly limited, and water,various buffers, and the like can be used, for example. Examples of thebuffers include Tris-HCl, sodium phosphate, EPPS, HEPES, and TESbuffers. Among these, Tris-HCl and sodium phosphate buffers arepreferable. Furthermore, the concentration of the buffer is, forexample, in the range from 10 to 300 mmol/l, preferably from 50 to 300mmol/l.

The surfactant is not particularly limited, and may be, for example,polyoxyethylene alkyl ether such as Brij 35, Brij 58, andpolyoxyethylene lauryl ether, polyoxyethylene alkylphenyl ether such asTriton X-100 and Triton X-114, polyoxyethylene sorbitan ether such asTween 20 and Tween 60, and the like.

The tetrazolium compound preferably has substituents with a ringstructure (ring substituents) at least at two positions on its tetrazolering, more preferably at three positions on its tetrazole ring, forexample.

In the case where the tetrazolium compound has ring substituents atleast at two positions on its tetrazole ring as described above, it ispreferable that the ring substituents are at the 2-position and3-position on the tetrazole ring. Further, in the case where thetetrazolium compound has ring substituents at three positions on itstetrazole ring, it is preferable that the ring substituents are at the2-position, 3-position, and 5-position on the tetrazole ring.

Further, it is preferable that at least two ring substituents of thetetrazolium compound have a benzene ring structure. Other than thebenzene ring structure, the ring substituents may have a resonancestructure with S or O being contained in the ring skeleton, for example.Examples of the ring substituents with such a resonance structureinclude a thienyl group, thiazoyl group, and the like.

Furthermore, it is preferable that the tetrazolium compound has ringsubstituents at least at three positions on its tetrazole ring and atleast two of the ring substituents have a benzene ring structure.

Still further, it is preferable that at least one ring substituent has afunctional group, and a larger number of functional groups are morepreferable.

Preferable examples of the functional group include electron-withdrawingfunctional groups such as a halogen group, ether group, ester group,carboxy group, acyl group, nitroso group, nitro group, hydroxy group,and sulfo group. Examples other than these functional groups includecharacteristic groups containing oxygen such as a hydroperoxy group, oxygroup, epoxy group, epidioxy group, and oxo group; and characteristicgroups containing sulfur such as a mercapto group, alkylthio group,methylthiomethyl group, thioxo group, sulfino group, benzenesulfonylgroup, phenylsulfonyl group, p-toluenesulfonyl group, p-tolylsulfonylgroup, tosyl group, sulfamoyl group, and isothiocyanate group. Amongthese electron-withdrawing functional groups, a nitro group, sulfogroup, halogen group, carboxy group, hydroxy group, methoxy group,ethoxy group are preferable. Examples other than the above-describedelectron-withdrawing functional groups include unsaturated hydrocarbongroups such as a phenyl group (C₆H₅—) and styryl group (C₆H₅CH═CH—). Itis to be noted that the functional groups may have been ionized bydissociation.

Still further, it is preferable that the tetrazolium compound hasbenzene rings at the 2-position and 3-position on its tetrazole ring andat least one of the benzene rings has at least one functional groupselected from the group consisting of a halogen group, carboxy group,nitro group, hydroxy group, sulfo group, methoxy group, and ethoxygroup. It is to be noted here that both the benzene rings may have sucha functional group. Further, the functional group may be at anypositions (ortho-, meta-, pra-) on each of the benzene rings.Furthermore, the number of the functional groups is not particularlylimited, and the benzene ring may have either the same or differentfunctional groups.

Examples of the tetrazolium compound having ring substituents with abenzene ring structure at the 2-position, 3-position, and 5-position onits tetrazole ring include:

-   2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium    salt;-   2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium    salt;-   2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium    salt;-   2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium salt;-   3,3′-(1,1′-biphenyl-4,4′-diyl)-bis(2,5-diphenyl)-2H-tetrazolium    salt;-   3,3′-[3,3′-dimethoxy-(1,1′-biphenyl)-4,4′-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium    salt];-   2,3-diphenyl-5-(4-chlorophenyl)tetrazolium salt;-   2,5-diphenyl-3-(p-diphenyl)tetrazolium salt;-   2,3-diphenyl-5-(p-diphenyl)tetrazolium salt;-   2,5-diphenyl-3-(4-styrylphenyl)tetrazolium salt;-   2,5-diphenyl-3-(m-tolyl)tetrazolium salt; and-   2,5-diphenyl-3-(p-tolyl)tetrazolium salt.

The tetrazolium compound is not limited to those described above. Inaddition to the above-described tetrazolium compounds, tetrazoliumcompounds having ring substituents with a benzene ring structure at twopositions and one ring substituent with a structure other than thebenzene ring structure at one position on its tetrazole ring also may beused. Examples of such tetrazolium compounds include:

-   2,3-diphenyl-5-(2-thienyl)tetrazolium salt;-   2-benzothiazoyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethyl    carbamoyl)phenyl]-2H-tetrazolium salt;-   2,2′-dibenzothiazoyl-5,5′-bis[4-di(2-sulfoethyl)carbamoylphenyl]-3,3′-(3,3′-dimethoxy-4,4′-biphenylene)ditetrazolium    salt; and-   3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium salt.

Further, tetrazolium compounds having ring substituents with a benzenering structure at two positions and one substituent not having a ringstructure at one position on its tetrazole ring also can be used.Examples of such tetrazolium compounds include:

-   2,3-diphenyl-5-cyano tetrazolium salt;-   2,3-diphenyl-5-carboxy tetrazolium salt;-   2,3-diphenyl-5-methyltetrazolium salt; and-   2,3-diphenyl-5-ethyl tetrazolium salt.

Among the above-described tetrazolium compounds, preferable are thosehaving three ring substituents as described above, and more preferableare those having three ring substituents with a benzene ring structureand having many electron-withdrawing functional groups. Particularlypreferable is2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt. It is to be noted here that the above-described tetrazoliumcompounds may be salts or may have been ionized, for example. Moreover,the tetrazolium compound may be used either alone or in combinations oftwo or more types.

The solution prepared so as to prevent the erroneous color developmentof DA-64 as described above can be used as a DA-64 reagent solution. Theapplication of the DA-64 reagent solution is not particularly limited.For example, it can be used as a color-developing substrate reagentsolution in a redox reaction described later, for example.

Hereinafter, the measurement method using a redox reaction according tothe present invention will be described in detail with reference to thefollowing example, in which a glycated protein in blood cells ismeasured.

First, whole blood itself is hemolyzed, or a blood cell fraction isseparated from whole blood in the usual way such as centrifugation andthen hemolyzed, so as to prepare a hemolyzed sample. The method ofcausing the hemolysis is not particularly limited, and can be, forexample, a method using a surfactant, a method using ultrasonic waves,and a method utilizing the difference in osmotic pressure. Among these,the method using a surfactant is preferable because of its simplicity inoperation, etc.

As the surfactant, for example, non-ionic surfactants such aspolyoxyethylene-p-t-octylphenyl ether (e.g. Triton series surfactants),polyoxyethylene sorbitan alkyl ester (e.g. Tween series surfactants),polyoxyethylene alkyl ether (e.g. Brij series surfactants), and the likecan be used. Specific examples are Triton X-100, Tween-20, Brij 35, andthe like. The conditions of the treatment with the surfactant usuallyare as follows: when the concentration of blood cells in the solution tobe treated is in the range from 1 to 10 vol %, the surfactant is addedso that its concentration in the solution falls in the range from 0.01to 5 wt %, and stirred at room temperature for about several seconds(about 5 seconds) to 10 minutes.

Next, a surfactant is added to the hemolyzed sample. In the case wherethe hemolyzed sample was prepared by causing the hemolysis using thesurfactant as described above, it is not necessary to further add thesurfactant if the concentration of the surfactant in the sample alreadyis within the following range. On the other hand, if the concentrationof the surfactant does not reach the following range, the surfactant maybe added so as to make up a shortfall.

The surfactant may be added so that its final concentration in areaction solution of a redox reaction described later falls in the rangefrom 0.006 to 80 mmol/l, preferably from 0.05 to 40 mmol/l, andparticularly preferably from 0.2 to 20 mmol/l. Furthermore, in thehemolyzed sample to which the surfactant has been added, theconcentration of the surfactant is, for example, in the range from 0.1to 200 mmol/l, preferably from 0.5 to 100 mmol/l, and particularlypreferably from 2 to 100 mmol/l.

Subsequently, a tetrazolium compound and sodium azide further are addedto the hemolyzed sample containing the surfactant.

The tetrazolium compound and the sodium azide may be added so that theirfinal concentrations in the reaction solution of the redox reaction fallwithin the following ranges: the tetrazolium compound in the range from0.01 to 40 mmol/l and the sodium azide in the range from 0.015 to 20mmol/l; preferably, the tetrazolium compound in the range from 0.1 to 32mmol/l and the sodium azide in the range from 0.05 to 12 mmol/l; andparticularly preferably, the tetrazolium compound in the range from 0.15to 24 mmol/l and the sodium azide in the range from 0.05 to 8 mmol/l.

Furthermore, the tetrazolium compound (A) and the sodium azide (B) areadded so that they are present at a ratio (molar ratio A:B), forexample, in the range from 10:1 to 1:1, preferably from 6:1 to 1.5:1,and more preferably from 4:1 to 2:1.

Specifically, when the concentration of blood cells in the solution tobe treated is in the range from 1 to 10 vol %, for example, thetetrazolium compound preferably is added so that its concentration inthe solution falls in the range from 0.02 to 2000 mmol/l, morepreferably from 0.1 to 1000 mmol/l, and particularly preferably from 0.4to 200 mmol/l. On the other hand, when the concentration of blood cellsin the solution to be treated is in the range from 1 to 10 vol %, forexample, the sodium azide preferably is added so that its concentrationin the solution falls in the range from 0.006 to 800 mmol/l, morepreferably from 0.04 to 400 mmol/l, and particularly preferably from 0.1to 80 mmol/l.

The tetrazolium compound and the sodium azide may be added to thehemolyzed sample simply as they are. However, in terms of simplicity inoperation etc., it is preferable to use a tetrazolium compound solutionobtained by dissolving the tetrazolium compound in a solvent and asodium azide solution obtained by dissolving the sodium azide in asolvent, or to use a liquid mixture containing both the tetrazoliumcompound and sodium azide (i.e., a tetrazolium compound-sodium azideliquid mixture).

As the solvent of the above-described solutions, MOPS, CHES, Tris-HCl,sodium phosphate, potassium phosphate, HEPES, TES buffers, and the likecan be used, for example. The pH of the solvent is, for example, in therange from 5 to 12, preferably from 6 to 10.

Moreover, the tetrazolium compound-sodium azide liquid mixture preparedpreferably is left for a certain period before being added to thehemolyzed sample so as to be aged, because this allows still furtherimprovement in sensitivity. According to this aging treatment, thesensitivity becomes, for example, about 1.2 to 3 times greater than inthe case where the aging treatment is not performed.

In the aging treatment, the treatment temperature preferably is in therange from 4° C. to 80° C., more preferably from 25° C. to 75°, andparticularly preferably from 40° C. to 700, and the treatment period is,for example, in the range from 10 minutes to 200 hours, more preferablyfrom 1 to 180 hours, and particularly preferably from 3 to 100 hours.

After the tetrazolium compound and sodium azide are added to thehemolyzed sample simply as they are or as the above-described solution,the pretreatment of the hemolyzed sample usually is carried out byincubating the sample at 10° C. to 40° C. for 1 to 10 minutes. Bypretreating the sample with the tetrazolium compound, the influence ofreducing substances and the like contained in the sample on a redoxreaction can be eliminated, whereby the accuracy of measurement isimproved. As described above, the tetrazolium compound contributes tothe improvement in the accuracy of measurement by eliminating theinfluence of the reducing substances. In addition, when the tetrazoliumcompound is present with sodium azide, the measurement sensitivity alsois improved.

Next, the pretreated hemolyzed sample containing the tetrazoliumcompound and sodium azide is treated with a protease. This proteasetreatment is carried out so that a FAOD used in the subsequent treatmentcan act on the analyte more easily.

The type of the protease is not particularly limited, and for example,serine proteases, thiol proteases, metalloproteinases, and the like canbe used. Specifically, trypsin, proteinase K, chymotrypsin, papain,bromelain, subtilisin, elastase, aminopeptidase, and the like arepreferable. In the case where the glycated protein to be degraded isglycated hemoglobin, the protease is the one that degrades the glycatedhemoglobin selectively, and bromelain, papain, trypsin derived fromporcine pancreas, metalloproteinases, and protease derived from Bacillussubtilis, and the like are preferable. Examples of the protease derivedfrom Bacillus subtilis include a product named Protease N (e.g., FlukaChemie AG) and a product named Protease N “AMANO” (Amano Enzyme Inc.).Examples of the metalloproteinases include metalloproteinase (EC 3. 4.24. 4) derived from the genus Bacillus. Among these, metalloproteinases,bromelain, and papain are more preferable, and metalloproteinases areparticularly preferable. Thus, a degradation product of a specificprotein can be prepared selectively by using a protease that degradesthe protein selectively. The protease treatment usually is carried outin a buffer, and the conditions of the treatment are determined asappropriate depending on the type of the protease used, the type and theconcentration of the glycated protein as an analyte, etc.

As the buffer, CHES, CAPSO, CAPS, phosphate, Tris, EPPS, HEPES buffers,and the like can be used, for example. The pH of the buffer is, forexample, in the range from 6 to 13, preferably from 7 to 10. Moreover,the final concentration of the buffer in the solution subjected to theprotease treatment is, for example, in the range from 1 to 200 mmol/l.

Specifically, when the pretreated hemolyzed sample is treated using ametalloproteinase as the protease, the protease treatment usually iscarried out under the conditions as follows: the concentration of themetalloproteinase in the reaction solution in the range from 2 to 20,000KU/I; the concentration of blood cells in the reaction solution in therange from 0.05 to 15 vol %; the reaction temperature in the range from15° C. to 37° C.; the reaction period in the range from 1 minute to 24hours; and the pH in the range from 6 to 12.

Furthermore, when the pretreated hemolyzed sample is treated usingproteinase K as the protease, the protease treatment usually is carriedout under the conditions as follows: the concentration of the proteasein the reaction solution in the range from 1 to 10,000 KU/1; theconcentration of blood cells in the reaction solution in the range from0.05 to 15 vol %; the reaction temperature in the range from 15° C. to37° C.; the reaction period in the range from 1 minute to 24 hours; andthe pH in the range from 6 to 12. Moreover, the type of the buffer isnot particularly limited, and for example, Tris-HCl, EPPS, PIPESbuffers, and the like can be used.

Next, the degradation product obtained by the protease treatment istreated with the FAOD. The reaction shown by Formula (1) above iscatalyzed by this FAOD treatment.

Similarly to the above-described protease treatment, this FAOD treatmentpreferably is carried out in a buffer. The conditions of the FAODtreatment are determined as appropriate depending on the type of theFAOD used, the type and the concentration of the glycated protein as ananalyte, etc.

Specifically, the FAOD treatment is carried out, for example, under theconditions as follows: the concentration of the FAOD in the reactionsolution in the range from 50 to 50,000 U/1, the concentration of theblood cells in the reaction solution in the range from 0.01 to 1 vol %,the reaction temperature in the range from 15° C. to 37° C., thereaction period in the range from 1 to 60 minutes, and the pH in therange from 6 to 9. Moreover, the type of the buffer is not particularlylimited, and the same buffers as in the protease treatment also can beused in the FAOD treatment.

Next, the hydrogen peroxide formed by the FAOD treatment is measured bya redox reaction using a POD and DA-64.

The DA-64 as a color-developing substrate may be added so that its finalconcentration in the reaction solution of the redox reaction falls inthe range from 0.001 to 20 mmol/l, preferably from 0.005 to 2 mmol/l,and particularly preferably from 0.01 to 0.5 mmol/l.

The DA-64 (C) and the already added surfactant (D), tetrazolium compound(E), and sodium azide (F) are present at a ratio (molar ratio C:D:E:F),for example, in the range from 1:6:10:3 to 1:2000:1000:500, preferablyfrom 1:10:20:10 to 1:1000:800:300, and more preferably from 1:40:30:10to 1:500:600:200.

The pH of this reaction solution is in the range from 6 to 9, preferablyfrom 6 to 8.

The redox reaction usually is carried out in a buffer. The conditions ofthe reaction are determined as appropriate depending on theconcentration of the hydrogen peroxide formed, etc. Specifically, theconditions are, for example, as follows: the concentration of the POD inthe reaction solution in the range from 10 to 100,000 IU/1; theconcentration of the color-developing substrate in the range from 0.001to 20 mmol/l; the reaction temperature in the range from 15° C. to 37°C.; the reaction period in the range from 0.1 to 30 minutes; and the pHin the range from 6 to 8. Moreover, the type of the buffer is notparticularly limited, and for example, the same buffers as in theprotease treatment and the FAOD treatment can be used.

The DA-64 develops color by a redox reaction. Thus, by measuring theabsorbance (i.e., the degree of the color developed) of the reactionsolution with a spectrophotometer at a wavelength, for example, in therange from 650 to 760 nm, the amount of the hydrogen peroxide can bedetermined. Then, using the amount of the hydrogen peroxide thusdetermined and a previously prepared calibration curve showing thecorrelation between an amount of hydrogen peroxide and an amount ofglycated protein, the amount of the glycated protein in the sample canbe determined.

Thus, by adding a surfactant in addition to a tetrazolium compound andsodium azide and setting the ratio of these components in theabove-described range, the erroneous color development of DA-64 can beprevented. As a result, an increase in background can be suppressed sothat high measurement accuracy can be realized.

Furthermore, as described above, the analyte is not particularly limitedas long as a redox reaction is utilized. Examples of the analyte otherthan the glycated protein include glycated peptides, glycated aminoacids, glucose, cholesterol, uric acid, creatinine, sarcosine, andglycerol. When the amount of each of the above-described examples of theanalyte is measured, measurement can be carried out, for example, byforming an oxidizing substance derived from the analyte and measuringthe amount of the oxidizing substance using a redox reaction in the samemanner as described above.

For example, when the measurement is carried out by forming hydrogenperoxide as an oxidizing substance derived from the analyte, thehydrogen peroxide may be formed, for example, by action of: a glucoseoxidase on the glucose; a cholesterol oxidase on the cholesterol; auricase on the uric acid; a sarcosine oxidase on the creatinine; asarcosine oxidase on the sarcosine; or a glycerol oxidase on theglycerol; respectively. The amount of the hydrogen peroxide can bemeasured in the same manner as above. Moreover, glycated peptides andglycated amino acids can be measured, for example, in the same manner asin the measurement of the glycated protein described above.

EXAMPLES

(Treatment Solution A)

-   A-1: 40 mmol/l CHES buffer (pH 9.5) containing 7 g/l (12 mmol/l) of    polyoxyethylene (9) lauryl ether (PEGLE)-   A-2: 40 mmol/l CHES buffer (pH 9.5) containing 50 g/l (85 mmol/l) of    PEGLE    (Treatment Solution B)

A mixture containing 5 mmol/l of2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt (product name WST3, manufactured by Dojindo Laboratories) and 1.5mmol/i of sodium azide was prepared and incubated at 50° C. for 24hours. Then, 3 ml of this mixture, 1 ml of a metalloproteinase (ARKRAY,Inc., 10,000 KU/1), 1 ml of MES buffer (50 mmol/l, pH 5.5), NaClsolution (500 mmol/l), and 3 ml of purified water were mixed with eachother. The resultant mixture was used as a treatment solution B.

(Treatment Solution C) C-1: DA-64 80 μmol/l Tris buffer (pH 7.0) 300mmol/l FAOD (ARKRAY, INC.) 30 KU/l POD 100 KU/l C-2: DA-64 1000 μmol/lTris buffer (pH 7.0) 300 mmol/l FAOD (ARKRAY, INC.) 30 KU/l POD 100 KU/l(Procedure)

Blood was centrifuged (3000 rpm), and blood cells were collected. Then,10 μl of the blood cells were mixed with 300 μl of the treatmentsolutions A (A-1 and A-2), respectively, to prepare hemolyzed samples.Then, 100 μl of the treatment solution B was added to 10 μl of thesehemolyzed samples, and the resultant mixtures were incubated at 37° C.for 5 minutes. Subsequently, 22 μl of the treatment solutions C(C-1 andC-2) respectively were added to each of the mixtures, and the resultantmixtures were incubated at 37° C. The absorbance (at the wavelength of751 nm) of these reaction solutions was measured using an automaticanalysis apparatus named JCA-BM 8 (manufactured by Japan Electron OpticsLaboratory Co. Ltd.). The instant at which the hemolyzed samples weremixed with the treatment reagent B was regarded as 0 second, and theinstant at which the treatment solutions C were added was 300 seconds.In order to measure the erroneous color development occurringcontinuously after the completion of the reaction between the FAOD andthe POD, the amount of change in absorbance between 486 seconds and 603seconds was determined. Table 1 below shows the final concentrations ofthe respective components in the reaction solutions and the amount ofthe respective components relative to 1 μmol of the DA-64. Furthermore,FIG. 1 and Table 2 below show the results of the determination of theamount of the change in absorbance. FIG. 1 is a graph showing the changein absorbance of the reaction solution with time.

TABLE 1 Ex. 1 Com. Ex. 1 Com. Ex. 2 Com. Ex. 3 Treatment solution A A-1A-2 A-1 A-2 Treatment solution B B B B B Treatment solution C C-1 C-1C-2 C-2 Final concentration during reaction PEGLE (mmol/l) 0.909 6.440.909 6.44 WST-3 (mmol/l) 1.136 1.136 1.136 1.136 NaN₃ (mmol/l) 0.3410.341 0.341 0.341 DA-64 (μmol/l) 13.3 13.3 166.7 166.7 Amount ofrespective components PEGLE (mmol/l) 0.068 0.483 0.005 0.039 WST-3(mmol/l) 0.085 0.085 0.0068 0.0068 NaN₃ (mmol/l) 0.026 0.026 0.00200.0020 DA-64 (μmol/l) 1.0 1.0 1.0 1.0

TABLE 2 Ex. 1 Com. Ex. 1 Com. Ex. 2 Com. Ex. 3 Amount of change 0.00490.0091 0.0087 0.0099 in absorbance

As shown in Table 1 above, in Comparative Example 1, the amount of thePEGLE was too large (>0.4 mmol) relative to 1 μmol of the DA-64. InComparative Example 2, the amounts of the PEGLE (<0.006 mmol), the WST-3(<0.01 mmol), and the NaN₃ (<0.003 mmol) were too small relative to 1μmol of the DA-64, and in Comparative Example 3, the amounts of theWST-3 (<0.01 mmol) and the NaN₃ (<0.003 mmol) were too small relative to1 μmol of the DA-64. Thus, Comparative Examples 1 to 3 exhibited highabsorbance owing to the erroneous color development as shown in FIG. 1,and thus the amount of the change in absorbance was considerable asshown in Table 2. In contrast, in Example 1, since the erroneous colordevelopment was prevented, the absorbance was low as shown in FIG. 1 andthus the amount of the change in absorbance was reduced. These resultsdemonstrate that the method of the present invention can prevent theerroneous color development of DA-64, thereby improving the accuracy ofmeasurement.

INDUSTRIAL APPLICABILITY

As specifically described above, according to the present invention,erroneous color development of DA-64 as a color-developing substrate canbe prevented. Therefore, by applying the method of preventing theerroneous color development to measurement using a redox reaction, anincrease in background due to the erroneous color development can besuppressed so that the accuracy of the measurement can be improved.Moreover, by applying such a measurement method to, for example, themeasurement of HbA1c in erythrocytes, it becomes possible to realizemeasurement with higher accuracy than in conventional methods, whichfurther increases the importance of HbA1c as an indicator in thediagnosis and the like of diabetes.

1. A method of measuring an oxidizing substance derived from an analytein a sample, comprising: causing a reaction between the oxidizingsubstance derived from the analyte andN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt as a substrate that develops color as a result of thereaction, the reaction being caused by an oxidoreductase in the presenceof a tetrazolium compound and sodium azide, theN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt, the tetrazolium compound and the sodium azide being presentin an aqueous solution with a surfactant; and determining an amount ofthe oxidizing substance by measuring the color developed by thesubstrate, the method farther comprising, before the step of causing areaction, determining an amount of the tetrazolium compound, the sodiumazide, and the surfactant, and a pH of the solution, that will reduceerroneous color formation of theN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt as a result of the reaction, and mixing the tetrazoliumcompound and the sodium azide in the determined amounts with theN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt in the presence of the determined amount of the surfactantin an aqueous solvent, wherein the determined amounts are selected toprovide concentrations in the mixture in the ranges of 0.01 to 1 mmol ofthe tetrazolium compound, 0.003 to 0.5 mmol of the sodium azide, and0.006 to 0.4 mmol of the surfactant, per μmol of theN-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt, and the pH of the mixture is in a range of 6 to
 9. 2. Themethod according to claim 1, wherein the aqueous solvent is a samplesolution containing the analyte.
 3. The method according to claim 2,wherein the tetrazolium compound and the sodium azide arc added to thesample solution and thereafter, the N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylwnino)dipheaylamine sodium salt and theoxidoreductase further are added to the sample solution in the presenceof the surfactant to cause the reaction by the oxidoreductase.
 4. Themethod according to claim 3, wherein the analyte is treated with aprotease prior to the reaction caused by the oxidoreductase.
 5. Themethod according to claim 4, wherein the oxidoreductase is caused to acton a degradation product of the analyte obtained by the treatment withthe protease.
 6. The method according to claim 1, wherein theoxidoreductase is a fructosyl amino acid oxidase.
 7. The methodaccording to claim 1, wherein the oxidizing substance derived from theanalyte is hydrogen peroxide.
 8. The method according to claim 1,wherein the analyte is a glycated protein.
 9. The method according toclaim 8, wherein the analyte is glycated hemoglobin.
 10. The methodaccording to claim 1, wherein the tetrazolium compound is2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt.